Variable heavy chain only libraries, methods of preparation thereof, and uses thereof

ABSTRACT

The present disclosure relates to designs and applications of Variable Heavy Only (VHO) domain regions. The present disclosure provides a VHO library of polynucleotides encoding VHO domains, wherein the VHO domains are designed based on the Vh domain of a human Vh family, and a method of generating the VHO library. The present disclosure also provides a phage library displaying the VHO domains encoded by the VHO library through the use of M13 bacteriophage minor coat proteins such as pIX and pVII and a method of generating the phage library. The present disclosure also provides a method of screening the phage library to identify VHO candidates that are capable of binding a target of interest.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority of Provisional Application No.63/120,842, filed on Dec. 3, 2020, the content of which is incorporatedherein by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Dec. 2, 2021, isnamed 15271_0007-00000_SL and is 19,819 bytes in size.

FIELD OF THE DISCLOSURE

The present disclosure relates to designs and applications of VariableHeavy Only (VHO) domain regions, which can serve as selective bindingmoieties and can be used as therapeutic molecules and/or diagnosticreagents. The present disclosure also relates to methods of generatingdiverse VHO degenerate libraries, and methods of displaying degenerateVHO libraries using coat proteins of bacteriophages.

BACKGROUND OF THE DISCLOSURE

VHH (variable heavy homodimers) or single domain antibodies have beenstudied for some time and are being used in medicine (Muyldermans 2013);(Belanger, Iqbal et al. 2019). The camelid Heavy chain antibodies (HcAb)are found in Camelidae family (llama, camel, alpaca, etc.), and similarVariable New Antigen Receptor (VNAR) molecules are found inChondrichthyes and Cyclostomata classes that include sharks andlampreys. The VHH most studied and used recombinantly to date are thosefrom llamas and camels (Arbabi-Ghahroudi 2017). References made hereinto the camel/llama family refer to the members of the Camelidae family.The Camelidae family antibodies are heavy chain only molecules sized at70-90 kDa compared to human antibodies (IgG), which contain both heavyand light chains at 150-160 kDa in size (see FIG. 1A). The camel andllama antibodies consist, respectively, of 3 domains: variable domain,constant domain 2, and constant domain 3. The variable domain, like mostantibodies, has 3 hypervariable loops with 4 framework regions inbetween those hypervariable loops. The variable region of a camel orllama antibody is the site for binding to foreign proteins during ahumoral response. Like human antibodies, the diversity of paratope isdefined by the hypervariable regions or complimentary determiningregions (CDR) (Mitchell and Colwell 2018).

The Vh domains of camel and llama antibodies have been used to engineersingle domain and nanobody molecules (Arbabi-Ghahroudi 2017). Thesemolecules are ˜15 kDa in size and have higher thermal stability comparedto IgG (McConnell, Spasojevich et al. 2013). Because of thesebiophysical attributes, the single domain molecules provide an advantagefor manufacturing and development (Tonikian and Sidhu 2012) (Ewert,Cambillau et al. 2002) (Harmsen and De Haard 2007). These attributesalso provide advantages for more unique diagnostic and therapeuticapplications. They can be engineered to pass through cell membranes, tocross the blood brain barrier, and to serve as alternative biologicdelivery systems (Herce, Schumacher et al. 2017) (Bruce, Lopez-Islas etal. 2016). Examples of alternative drug delivery using single domainsinclude adapting them with nanoparticles for guided systemic delivery(Yang, Moynihan et al. 2018) (Slastnikova, Ulasov et al. 2018) andefficient intranasal delivery (Gomes, Cabrito et al. 2018). Thesemolecules can also be designed as fluorescent protein fusions to serveas imaging diagnostics (Li, Bourgeois et al. 2012). Some diseasetargets, e.g., viruses, possess size constraints for IgGs, and thereforesmaller protein therapeutics such as single domains are needed. (Wrapp,De VLieger et al. 2020) (Wilken and McPherson 2018).

The camel and llama antibody variable regions have protein sequencehomology to human Vh domains (Mitchell and Colwell 2018), (Herold, Johnet al. 2017), (Muyldermans 2013), (Strohl et al., (2012), WoodheadPublishing). FIG. 1A shows the structures of human IgG and camelidantibodies. The consensus translations of the V gene segments for human,llama, and camel are aligned in FIG. 1(B) showing their amino acidsequence similarity (SEQ ID NO: 1-4). CDR3 is not shown in FIG. 1(B)because that is where the most diversity of an antibody resides. FIG. 2shows a tree-based alignment of human Vh families to both camel andllama Vh families obtained from IMGT (the international ImMunoGeneTicsinformation system)(http://www.imgt.org/IMGTrepertoire/Proteins/taballeles/human/IGH/IGHV/Hu_IGHVall.html).The grouping shows the human family Vh3 to be most similar in sequencehomology to both camel and llama VH, but not so much with the otherhuman Vh families. FIG. 3 shows further sequence homology within thehuman Vh3 sub-families.

The human Vh3 (also referred to as Vh3) family is the most prevalent onefound during a human humoral response when immunized, as well asreflective of the distribution of antibodies in development (Joyce,Burton et al. 2020) (Longo, Rogosch et al. 2017). This family is alsofound to be the most represented in the human repertoire (Tiller,Schuster et al. 2013). Even though camel/llama single Vh domains aresimilar to human Vh domains of antibodies, they are also quite distinct.These differences may create immunogenic effects that limit the efficacyof diagnostic and therapeutic treatments using camel/llama single Vhdomains.

Certain researchers have based their phage libraries from camel, llama,or alpaca antibody scaffolds. Lately, Twist Bioscience used scaffoldsbased on a mix of sequences of human and one of the other species(camel, llama, and alpaca). The sdAb (single domain antibodies) reviewby Rossotti et al. (2021) concluded that immunogenicity and anti-drugantibody responses (ADA) to sdAbs occurred from the intrinsic non-humansequences that resulted in the aggregation properties.

The present disclosure describes using human Vh regions in place ofCamelid-derived VHH-based molecules, to generate single domain-baseddiagnostic or therapeutic compounds. For example, the present disclosuredescribes using the Vh regions of the human antibody Vh3 family togenerate single domain-based therapeutic compounds. Such singledomain-based therapeutic compounds may have improved stability and/orimproved therapeutic index than single domain-based therapeuticcompounds derived from other human antibody Vh families.

SUMMARY OF THE DISCLOSURE

The present disclosure provides a VHO (variable heavy only) platform togenerate single domain-based therapeutic compounds, e.g., sdAb,nanobodies (Nb), and VHH type of antibodies. In one embodiment, thepresent disclosure provides a VHO library (also referred to herein as aTavoSelect library) based on the human variable domain of a human Vhfamily, such as the gene family Vh3. For example, the Vh regions of thehuman antibody Vh3 family can be used as a scaffold for creating VHOlibraries.

In one embodiment, the present disclosure provides a VHO library basedon human variable domain germlines that are homologous by amino acidsequence and/or similar in canonical structure to the Vh domains of ahuman Vh family. In one embodiment, the present disclosure provides aVHO library of polynucleotides encoding diverse VHO domains, wherein theVHO domains have sequence homology and/or canonical homology with the Vhdomain of a human Vh family, such as the gene family Vh3. For example, acanonical structure can include 3D modeling of human antibody sequencesfound at the IMGT (the international ImMunoGeneTics information system)or any other source such as Kabat database or V Base database.

In one embodiment, the present disclosure provides a phage library thatis capable of displaying diverse VHO domains or displays diverse VHOdomains (herein referred to as “a VHO phage library”). In oneembodiment, a VHO library is assembled on a coat protein on a bacterialphage, such as M13 phage. In one embodiment, a VHO library is assembledby way of genetic fusion to the pIX and/or pVII coat protein of an M13phage.

In one embodiment, the present disclosure provides a VHO phage librarythat is sufficiently robust to undertake a diverse selection or panningscreening.

In one embodiment, the present disclosure provides a vector comprising apolynucleotide encoding any one of the VHO domains described herein.

In one embodiment, the present disclosure provides a method of preparinga VHO library, comprising providing polynucleotide sequences encodingthe VHO domains described herein, and inserting the polynucleotidesequences into a vector, such as a phagemid and/or a plasmid.

In one embodiment, the present disclosure provides a method of preparinga VHO phage library, comprising transforming a bacterial cell culturewith a VHO library described herein, allowing the bacterial cell cultureto grow to a log phase, infecting the bacterial cell culture with ahelper phage, and amplifying the bacterial cell culture.

In one embodiment, the present disclosure provides a method of screeninga VHO phage library, e.g., by phage panning, to identify a VHO candidateof interest. In one embodiment, the present disclosure provides a methodof identifying a VHO domain of interest that is capable of binding atarget, comprising: creating a VHO library such as a VHO phage libraryas described herein, screening the VHO phage library using biopanningagainst the target to identify the VHO of interest. In one embodiment,the method further comprises sequencing the VHO of interest by NGS (nextgeneration sequencing). In one embodiment, the method further comprisesevaluating the binding affinity of the VHO of interest to the target,e.g., by ELISA, BLI, and/or SPR.

In one embodiment, VHO candidates of interest can be presented on aphage, expressed as a stand-alone protein, expressed as a fusion with anIgG or another soluble domain, or as a protein fusion to a cell surfaceprotein such as a T or B cell receptor domain.

In one embodiment, a VHO as described herein can be connected to a humanIgG Fc domain as a fusion. Such a fusion to an Fc domain may or may notpossess a hinge region, or may or may not be fused to a constant heavychain domain, or may or may not be fused to a constant light chain,either of the kappa or lambda families.

In one embodiment, the present disclosure provides a VHO of interestthat can bind a target. In one embodiment, the present disclosureprovides a protein that has greater than 50% identity to a VHO ofinterest. In one embodiment, the present disclosure provides apolynucleotide that encodes a protein that has greater than 50% identityto a VHO of interest.

In one embodiment, a VHO of interest is fused to a tag. The tag can bechosen from an Fc domain of any immunoglobulin family, poly histidine,and FLAG. For example, a VHO of interest is fused to a protein chosenfrom immunoglobulins, receptors, cell surface proteins, and fragments ofthe immunoglobulins, receptors, and cell surface proteins. For example,the VHO of interest can be expressed as a soluble protein, as a solubleprotein fused to another protein (e.g., immunoglobulins), or as a fusionto any cell surface protein (e.g., receptors) in any bacterial cell(e.g., E. coli), any mammalian cell, yeast, or plant cell.

In one embodiment, the present disclosure provides a compositioncomprising a VHO of interest or a polypeptide comprising the VHO ofinterest, e.g., a fusion of the VHO of interest with another protein.

In one embodiment, the present disclosure provides a cell, e.g., abacterial cell, comprising a vector as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

This patent or application file contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A shows structural models portraying an IgG and Camelid HcAb with3D ribbon structures representing the variable heavy domains, denoted asVh and VHH, respectively. FIG. 1B shows a consensus sequence (SEQ IDNO: 1) aligned with the Vh domains of human (SEQ ID NO: 2), camel (SEQID NO: 3), and llama (SEQ ID NO: 4) antibodies including regions FR1,CDR1 (underlined), FR2, CDR2 (underlined), and FR3 according to IMGTnomenclature. The antibody images depicted on FIG. 1A can be found athttps://www.frontiersin.org/files/Articles/288027/fimmu-08-00977—HTML/image_m/fimmu-08-00977-g001.jpg.CDR3 is not shown in FIG. 1B.

FIG. 2 shows a homology amino acid sequence alignment tree generatedusing the Geneious Prime software (https://www.geneious.com/prime/)using the amino acid sequences of the human, llama, and camel heavyV-region families from the IMGT.

FIG. 3 shows homology amino acid sequence alignment trees generatedusing the Geneious Prime software (https://www.geneious.com/prime/).FIG. 3A shows amino acid sequence alignment of the human Vh3sub-families and llama Vh families. FIG. 3B shows amino acid sequencealignment of the human Vh3 subfamilies and camelid Vh families.

FIG. 4A is a schematic drawing of an M13 filamentous bacteriophage andFIG. 4B illustrates the coat proteins that make up the exterior of anM13 phage.

FIG. 5 illustrates components that make up a VHO phage library. FIG. 5Ais a schematic representation of a gene cassette showing DNA segmentsencoding a VHO fused to pIX (a coat protein of M13 phage), HIS(hexa-histidine residue (SEQ ID NO: 12)), and HA (hemagglutinin). FIG.5B illustrates a phagemid/plasmid (herein referred to as pTAVOphagemid/plasmid), wherein a VHO gene cassette can be spliced into thepTAVO phagemid/plasmid in between the NcoI and NotI restriction sites.This phagemid/plasmid can be used for VHO gene expression and/or phagedisplay libraries. FIG. 5C is a representative rendition of a VHO fusedor displayed on the surface of an M13 phage.

FIG. 6 shows a tabulated distribution of 55 VHO candidates from a VHOphage library panned against three orthologs (human, cynomolgus monkey,and mouse) of the target, TNF (tumor necrosis factor). Each section(under “Human panning,” “Cyno panning,” and “Mouse panning,”respectively) of the table in FIG. 6 shows a group of VHO candidatespanned against the three respective ortholog targets (human, cynocynomolgus monkey, and mouse TNF). Listed from the most copy sequencenumber to the least are the top 19 VHO candidates corresponding to therespective ortholog panning group. Within each section of a panninggroup are VHO sequences that were found in the other ortholog panninggroups, thus indicating cross-reactivity of VHOs with the respectiveortholog targets (e.g., VHO candidate Nos. 8 and 11 appear in both the“Human panning” group and the “Cyno panning” group, indicating VHO Nos.8 and 11 may have cross-reactivity with human and cyno TNF). Cyno is anabbreviation of cynomolgus monkey.

FIG. 7 shows biolayer interferometry (GatorBio) binding kinetic runsperformed with a set of VHO-Fc fusion molecules (VHO3-Fc, VHO4-Fc, andVHO5-Fc, comprising SEQ ID NOs: 7, 8, and 9 respectively), which werereactive to both human and mouse soluble FCRN proteins but not tobeta-2-microglobulin (B2M). Odd numbered channels (denoted as CH1, CH3,CH5, and CH7) were run at pH 7 and the even numbered channels (denotedas CH2, CH4, CH6, and CH8) were run at pH 6. The biolayer interferometrybinding sequence steps included (1)—binding of biotinylated FCRN (humanand mouse) or B2M to the streptavidin coated sensor probe, (2)—washingto allow for baseline buffer exchanges, (3)—association of VHO-Fcanalytes to the BLI sensor probe, (4)—dissociation of VHO-Fc analytesfrom FCRN or B2M on the BLI sensor probe. FIG. 7A showing VHO-Fc bindingto human FcRn; FIG. 7B showing VHO-Fc binding to murine FcRn; FIG. 7Cshowing VHO-Fc not binding to beta-2 macroglobulin. On each of FIGS. 7A,7B, and 7C, VHO3-Fc (comprising SEQ ID NO: 7) corresponds to CH1 andCH2; VHO4-Fc (comprising SEQ ID NO: 8) corresponds to CH3 and CH4;VHO5-Fc (comprising SEQ ID NO: 9) corresponds to CH5 and CH6; an IgG1corresponds to CH7, and a negative control VHO2-Fc (comprising SEQ IDNO: 11) corresponds to CH8.

FIG. 8A shows examples of size exclusion chromatography results of twodifferent VHO-Fc molecules (VHO1-Fc comprising SEQ ID NO: 10; VHO2-Fccomprising SEQ ID NO: 11) compared to a human IgG1 molecule. The resultsconfirm the presence of monodisperse VHO-Fc fusion proteins. FIG. 8Bshows an example of the purity of VHO-Fc proteins (Non reduced VHO-Fcfusion candidates after a magnetic bead-based purification) onnon-reduced SDS-PAGE.

FIG. 9 shows a chart of five target VHO panning experiment results goingthrough key steps of the VHO generation process. The first column of thetable describes a key behavior, epitope binning, or grouping. The VHOlibrary panning process can double the number of epitopes per number ofcandidates screened as compared to other sdAbs panning process in theart.

FIG. 10 shows an alignment of the closest human variable heavy domainsto the germline sub-family member (IGHV3-23). IGHV3-23 was used as thescaffold to build the VHO phage libraries in some embodiments of thisdisclosure. Recently, Wu et al (2020) describes generating an sdAb phagelibrary using the germline family IGHV3-66. The arrows point out someframework differences between IGHV3-66 and IGHV3-23. FIG. 10 disclosesSEQ ID NOs: 23-24, and 24-27, respectively, in order of appearance.

DETAILED DESCRIPTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though fully set forth. If certain content of a referencecited herein contradicts or is inconsistent with the present disclosure,the present disclosure controls.

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which the disclosure pertains.

Although any methods and materials similar or equivalent to thosedescribed herein may be used in the practice for testing of the presentdisclosure, exemplary materials and methods are described herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

“Antibodies” is meant in a broad sense and includes immunoglobulinmolecules including monoclonal antibodies including murine, human,humanized and chimeric monoclonal antibodies, antibody fragments,bispecific or multi-specific antibodies, dimeric, tetrameric, ormultimeric antibodies, single chain antibodies, domain antibodies andany other modified configuration of the immunoglobulin molecule thatcomprises an antigen binding site of the required specificity.

Full length antibody molecules are comprised of two heavy chains (HC)and two light chains (LC) inter-connected by disulfide bonds as well asmultimers thereof (e.g., IgM). Each heavy chain is comprised of a heavychain variable region (VH) and a heavy chain constant region (comprisedof domains CH1, hinge, CH2 and CH3). Each light chain is comprised of alight chain variable region (VL) and a light chain constant region (CL).The Vh and the VL regions may be further subdivided into regions ofhyper variability, termed complementarity determining regions (CDR),interspersed with framework regions (FR). Each Vh and VL is composed ofthree CDRs and four FR segments, arranged fromamino-to-carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2,FR3, CDR3 and FR4.

“Complementarity determining regions (CDR)” are “antigen binding sites”in an antibody. CDRs may be defined using various terms: (i)Complementarity Determining Regions (CDRs), three in the Vh (HCDR1,HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3), are based onsequence variability (Wu et al. (1970) J Exp Med 132: 211-50 (Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991).(ii) “Hypervariable regions,” “HVR,” or “HV,” three in the Vh (H1, H2,H3) and three in the VL (L1, L2, L3), refer to the regions of antibodyvariable domains which are hypervariable in structure as defined byChothia and Lesk (Chothia et al. (1987) J Mol Biol 196: 901-17. TheInternational ImMunoGeneTics (IMGT) database (http://www_imgt_org)provides a standardized numbering and definition of antigen bindingsites. The correspondence between CDRs, HVs and IMGT delineations isdescribed in (Lefranc et al. (2003) Dev Comp Immunol 27: 55-77. The term“CDR,” “HCDR1,” “HCDR2,” “HCDR3,” “LCDR1,” “LCDR2” and “LCDR3” as usedherein includes CDRs defined by any of the methods described supra,Kabat, Chothia or IMGT, unless otherwise explicitly stated in thespecification.

Conventional one and three-letter amino acid codes are used herein.Amino acid Three-letter code One-letter code:

Three-letter One-letter Amino acid code code Alanine Ala A Arginine ArgR Asparagine Asn N Aspartate Asp D Cysteine Cys C Glutamate Gln EGlutamine Glu Q Glycine Gly G Histidine His H Isoleucine Ile I LeucineLeu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro PSerine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine ValV

The polypeptides, nucleic acids, fusion proteins, and other compositionsprovided herein may encompass polypeptides, nucleic acids, fusionproteins, and the like that have a recited percent identity to an aminoacid sequence or DNA sequence provided herein. The term “identity”refers to a relationship between the sequences of two or morepolypeptide molecules or two or more nucleic acid molecules, asdetermined by aligning and comparing the sequences. “Percent identity,”“percent homology,” “sequence identity,” or “sequence homology” and thelike mean the percent of identical residues between the amino acids ornucleotides in the compared molecules and is calculated based on thesize of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) are preferably addressed by aparticular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), 1988, New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.In calculating percent identity, the sequences being compared aretypically aligned in a way that gives the largest match between thesequences.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein, and refer to a polymeric form of amino acids ofany length, which can include coded and non-coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified peptide backbones. The terms also includepolypeptides that have co-translational (e.g., signal peptide cleavage)and post-translational modifications of the polypeptide, such as, forexample, disulfide-bond formation, glycosylation, acetylation,phosphorylation, proteolytic cleavage, and the like.

Furthermore, as used herein, a “polypeptide” refers to a protein thatincludes modifications, such as deletions, additions, and substitutions(e.g., conservative in nature as would be known to a person in the art)to the native sequence, as long as the protein maintains a desiredactivity. These modifications can be deliberate, as throughsite-directed mutagenesis, or can be accidental, such as throughmutations of hosts that produce the proteins, or errors due to PCRamplification or other recombinant DNA methods.

The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and“nucleic acid molecule” are used interchangeably herein to include apolymeric form of nucleotides, either ribonucleotides ordeoxyribonucleotides. This term refers only to the primary structure ofthe molecule.

“Vector” refers to a polynucleotide capable of being duplicated within abiological system or that can be moved between such systems. Vectorpolynucleotides typically contain elements, such as origins ofreplication, polyadenylation signal or selection markers, that functionto facilitate the duplication or maintenance of these polynucleotides ina biological system, such as a cell, virus, animal, plant, andreconstituted biological systems utilizing biological components capableof duplicating a vector. The vector polynucleotide may be DNA or RNAmolecules, cDNA, or a hybrid of these, single stranded or doublestranded. The vector may be a bacterial phagemid and/or a plasmid. Thevector may be an expression vector or a vector that enables a phage todisplay a protein of interest. For example, a pTavo phagemid/plasmid asdisclosed herein can be a vector that comprises polynucleotides encodinga coat protein of a bacterial phage, or a vector that does not comprisepolynucleotides encoding such a coat protein. See, e.g., FIG. 5 (B). Thevector may comprise one or more tags, e.g., for purification and/ordetection, e.g., by ELISA, BLI, and/or SPR. The one or more tags can bechosen from poly-histidine (HIS) and hemagglutinin (HA) tags. The tagscan be fused with a VHO, and/or with the coat protein. See, e.g., FIG. 5(A).

“Expression vector” refers to a vector that can be utilized in abiological system or in a reconstituted biological system to direct thetranslation of a polypeptide encoded by a polynucleotide sequencepresent in the expression vector.

A “host cell,” as used herein, denotes an in vivo or in vitro eukaryoticcell or a cell from a multicellular organism (e.g., a cell line)cultured as a unicellular entity, which eukaryotic cells can be, or havebeen, used as recipients for a nucleic acid (e.g., an expression vectorthat comprises a nucleotide sequence encoding a polypeptide of thepresent disclosure), and include the progeny of the original cell whichhas been genetically modified by the nucleic acid. It is understood thatthe progeny of a single cell may not necessarily be completely identicalin morphology or in genomic or total DNA complement as the originalparent, due to natural, accidental, or deliberate mutation. A“recombinant host cell” (also referred to as a “genetically modifiedhost cell”) is a host cell into which has been introduced a heterologousnucleic acid, e.g., an expression vector. For example, a geneticallymodified eukaryotic host cell is genetically modified by virtue ofintroduction into a suitable eukaryotic host cell a heterologous nucleicacid, e.g., an exogenous nucleic acid that is foreign to the eukaryotichost cell, or a recombinant nucleic acid that is not normally found inthe eukaryotic host cell.

“Specific binding” or “specifically binds” or “binds” refer to anantibody binding to a specific antigen with greater affinity than foranother antigen. Typically, the antibody “specifically binds” when theequilibrium dissociation constant (KD) for binding is about 1×10-8 M orless, for example about 1×10-9 M or less, about 1×10-10 M or less, about1×10-11 M or less, or about 1×10-12 M or less, typically with the KDthat is at least one hundred-fold less than its KD for binding to anon-specific antigen (e.g., BSA, casein). The KD may be measured usingstandard procedures.

A “phage display library,” as used herein, refers to a proteinexpression library, constructed in a bacteriophage, e.g., anM13-derived, vector, which is capable of expressing or expresses acollection of cloned protein sequences as fusions with a phage coatprotein. Antibody phage display libraries, and methods of generatingsuch libraries, are known in the art (see, for example, Famm et al., J.Mol. Biol. 376:926-931, 2008; Carmen and Jermutus, Brief Funct GenomicProteomic 1(2):189-203, 2002; and U.S. Pat. Nos. 6,828,422 and7,195,866). For example, a VHO phage display library as disclosed hereinis capable of displaying or displays a library of VHO domains on aphage, e.g., an M13 phage.

“VHO,” “VHO region,” “VHO domain,” or “VHO protein,” as used herein,means a polypeptide that shares conserved sequences (protein or DNA),e.g., having at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% sequence identity, with the Vh region of human, camel,and llama antibody Vh families, e.g., the human Vh region of the humanantibody Vh3 family. Depending on the context, “VHO,” “VHO region,” or“VHO domain” may be used to refer to a polynucleotide sequence thatencode the VHO polypeptide.

Leader Peptides

In certain embodiments, a leader peptide or leader sequence is chosen todrive the secretion of a VHO protein described in this disclosure intothe cell culture supernatant as a secreted protein. Any leader peptidefor any known secreted proteins/peptides can be used.

As used herein, a “leader peptide” or “signal peptide” includes a shortpeptide, usually 16-30 amino acids in length, which is present at theN-terminus of newly synthesized proteins that are destined towards thesecretory pathway. Although leader peptides are extremely heterogeneousin sequence, and many prokaryotic and eukaryotic leader peptides arefunctionally interchangeable even between distinct species, theefficiency of protein secretion may be strongly determined by thesequence of the leader/signal peptide.

Linker Peptides

In some embodiments, there may be one or more linker peptides betweenthe VHO, the coat protein, and the one or more tags. See, e.g., FIG. 5(A). For example, there may be a linker peptide between the VHO and theone or more tags (e.g., Fc, HIS tag and/or HA tag). There may also be alinker peptide between VHO and the coat protein. In some embodiments,the linker peptide can be substituted by any other peptide that aids inthe activities for displaying on phage or use of tags for purificationor assay detection purposes.

Suitable linker peptides (also referred to as “spacers”) can be readilyselected, and can be of any of a number of suitable lengths, such asfrom 1 amino acid to 30 amino acids (e.g., any specific integer between1 and 30, or from 1 amino acid (e.g., Gly) to about 20 amino acids(e.g., 2-15, 3-12, 4-10, 5-9, 6-8, or 7-8 amino acids).

Exemplary linker peptides include glycine polymers (G)n, glycine-serinepolymers (including, for example, (GS)n, (GSGGS)n (SEQ ID NO: 13) and(GGGS)n (SEQ ID NO: 14), where n is an integer of at least one, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20),glycine-alanine polymers, alanine-serine polymers, alanine-proline,immunoglobulin isotype and subtype hinge that can comprise IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM, and other flexible linker peptides known inthe art. Both Gly and Ser are relatively unstructured, and therefore canserve as a neutral tether between components.

In certain embodiments, the linker peptide is a Glycine polymer. Glycineaccesses significantly more phi-psi space than even alanine and is muchless restricted than residues with longer side chains (see Scheraga,Rev. Computational Chem. 11173-142 (1992)). Exemplary linker peptidescan comprise amino acid sequences including, but not limited to, GGS,GGSG (SEQ ID NO: 15), GGSGG (SEQ ID NO: 16), GGGGS (SEQ ID NO: 17),GSGSG (SEQ ID NO: 18), GSGGG (SEQ ID NO: 19), GGGSG (SEQ ID NO: 20),GSSSG (SEQ ID NO: 21), and the like.

In certain embodiments, the linker peptide is a rigid linker (Chen, Zaroet al. 2013). Exemplary rigid linker peptides can comprise amino acidsequences including, but not limited to, proline-rich sequence, (XP)n,with X designating any amino acid, preferably Ala, Lys, or Glu, where nis an integer of at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20. Exemplary rigid linker peptides canalso comprise amino acid sequences including, but not limited to, alphahelix-forming linkers with the sequence of (EAAAK)n (SEQ ID NO: 22),where n is an integer of at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20.

Design, Synthesis, and Construction of VHO Libraries

The present disclosure provides a VHO platform to generate singledomain-based therapeutic compounds. In one embodiment, the presentdisclosure provides a VHO library (also referred to herein as aTavoSelect library) based on the human variable domain of a human Vhfamily, such as the gene family Vh3. The Vh regions of the humanantibody Vh3 family can be used as a scaffold for creating VHOlibraries. For example, the human sequence IGHV3.23, a member of theIGVh3 family, can be used as a scaffold to construct a VHO library. TheIGHV3 is selected because it has a more stable Vh than other humangermline families (Ewert et al 2003). IGHV3 also has a high level ofsequence identity to camel, llama, and alpaca IgG (see, e.g., FIGS. 2and 3).

The VHOs described herein differ from other single domain antibodies(sdAb) and/or nanobodies (Nb) in that the VHOs comprise human amino acidsequences. Thus, there may be no need for any humanization methods forVHO panning as there is for camel, llama, and alpaca sdAb libraries.

In one embodiment, a VHO library is created in such a way that itcaptures the highest accuracy possible. “Accuracy” is defined as theabsence of cysteine, methionine, and/or stop codons designed within theCDR regions of degeneracy as well as maintaining the open reading frame(ORF) from the initiating methionine codon to the designed stop codon.

In one embodiment, the present disclosure provides a VHO mutationallibrary constituting a library of VHO polynucleotide sequences withvariations (e.g., with randomized codons), wherein the VHO framework orscaffold is designed based on the Vh regions of the human Vh families,such as the Vh regions of the human Vh3 families. The Vh3 family, basedon IMGT nomenclature, may include the sub-family Vh3 members IGHV3-7,IVGH3-9, IVGH3-11, IVGH3-13, IVGH3-15, IVGH3-16, IVGH3-19, IVGH3-20,IVGH3-21, IVGH3-23, IVGH3-23D, IVGH3-25, IVGH3-30, IVGH3-30-3,IVGH3-30-5, IVGH3-33, IVGH3-35, IVGH3-38, IVGH3-43, IVGH3-47, IVGH3-48,IVGH3-49, IVGH3-52, IVGH3-53, IVGH3-64, IVGH3-66, IVGH3-69-1, IVGH3-72,IVGH3-73, and IVGH3-74.

In one embodiment, the present disclosure provides a VHO mutationallibrary constituting a library of VHO polynucleotide sequences withvariations (e.g., with randomized codons), wherein the VHO framework orscaffold is designed based on the Vh regions of IGHV3-23.

In one embodiment, the present disclosure provides a VHO library basedon human variable domain germlines that are homologous by amino acidsequence and/or similar in canonical structure to the Vh domains of ahuman Vh family. A canonical structure refers to the alignment of theCDR loops and the positioning of the sequences between the CDR loops.The human germline family Vh3 including all VDJ gene combinationsdefined by IMGT and/or other antibody resources are the closest insimilarity to llama and camel VHHs including their VDJ gene combinationsaccording to DNA and/or protein sequence BLAST search alignments basedon the IMGT and/or any other antibody resource. FIG. 3 shows that thesequences of camel or llama VHH domains compared to human Vh domainshave an average pairwise identity of 81% and 79% homology, respectively.The Vh3 germline family is most abundant in naïve and immunized humans(Herce, Schumacher et al. 2017). Vh3 germline family is more stable thanother human heavy variable germline families (Ewert, Huber et al. 2003).

In one embodiment, a VHO library is created based on the consensussequence of the fully human germline Vh3 family, e.g., generated byGeneious or other related software, by aligning all VDJ genecombinations as defined by the IMGT and/or other antibody resources. Thealignment of the Vh3 family sequences can be based on the nucleotide ornucleic acid sequences (DNA alignment) or amino acid sequences (proteinalignment). DNA sequences can also be generated corresponding to thealigned amino acid or residue type. Protein alignments can be performedapplying the rules of canonical structures provided by resources likeIMGT and the like.

In one embodiment, the present disclosure provides a VHO degenerate genelibrary of polynucleotides encoding various VHO (variable heavy only)domains, wherein the VHO domains have sequence homology and/or canonicalloop region homology defined by IMGT with the Vh domain of a human Vhfamily, such as the Vh3 family, e.g., IGHV3-23. For example, thesequence homology (amino acids or nucleotides) is at least 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. The VHO domains can haveresidue diversity throughout the entire regions of the VHO domains. Forexample, the VHO domains have residue diversity in one or more of theCDR regions, e.g., in CDR1, CDR2, and/or CDR3 regions. For example, theVHO domains have residue diversity in one or more of the frameworkregions, e.g., in FR1, FR2, FR3, and/or FR4 regions. In one embodiment,the one or more of the framework regions can have one or more mutationsthat provide improvements in levels of protein expression, proteinfolding, protein purification, binding affinity, downstream targetsignaling, and/or inhibition of signaling. In one embodiment, the VHOdomains have residue diversity in one or more of the CDR regions and inone or more of the framework regions. In one embodiment, the VHO domainshave residue diversity in all of the CDR regions and in all of theframework regions. In one embodiment, the VHO comprises regions, e.g.,CDR 1-3 and FR 1-4, typical of variable heavy chain domains according toIMGT or similar reference sources.

In one embodiment, a VHO library is designed to comprise gene modulesthat have CDR1 and CDR2 as one section and CDR3 as another section. TheCDRs can also be designed to minimize aggregation by disrupting residuesinvolved with the Vh and VL associations found in scFv, Fab, and IgGmolecules. Using different residues at certain positions in the CDRs aswell as the frameworks that normally provide Vh regions to interact withV1 regions, will reduce the VHO molecules from aggregating. Thus, theVHO library is different from the camel, llama, and alpaca VHH antibodylibraries where aggregation arises from intermolecular sdAbinteractions.

In one embodiment, the small paratopes of sdAbs derived from a VHOlibrary disclosed herein will enable screening of many formats ofbiopanning of a given target antigen. This diverse biopanning approachalong with the smaller paratopes of the VHOs enables many epitopes to bediscovered on the target surface. Having more epitopes can improve thechance of finding superior therapeutic or diagnostic biologicsmolecules.

In one embodiment, the length of CDR3, defined by either IMGT, KABAT,CLOTHIA, or Martin definitions as well as combinations of such, canrange from 1 to 30 amino acids, such as 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, to 30 aminoacids (6 to 30 codons of the corresponding polynucleotide sequence).

In one embodiment, a VHO library can include one or more sub libraries.For example, the length of CDR3 on the VHOs can be varied, thus multiplesub libraries within a VHO library can be created.

In one embodiment, the VHO framework or scaffold is designed based onthe following consensus sequences, generated by Geneious or otherrelated software uploaded with amino acid sequences from IMGT, of all VJregions of human Vh families:

(SEQ ID NO: 5) QVQLVESGGGLVKPGGSLRLSCAASGFTFS(X)WVRQAPGKGLEWV(X)DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR(X)WGQGTLVTVS S.

In one embodiment, the VHO framework or scaffold is designed based onthe following consensus sequences, generated by Geneious or otherrelated software uploaded with amino acid sequences from IMGT, of all VJregions of human Vh3 subfamilies:

(SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASGFTFS(X)NWVRQAPGKGLEWV(X)DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR(X)WGQGTLVTVS S.

Certain non-conserved positions in the CDR regions of a human variableheavy domain are marked by “X” in SEQ ID NOs: 5 and 6. The “X” is asymbol for degenerate mutagenesis of any kind applied to a codon that istranslated into appropriate amino acid. The placement of “X” can be setat any defined CDR positions according to IMGT, KABAT, Clothia, orMartin definitions as well as combinations of such definitions for humanvariable regions. An “X” can be placed at any position within a variableheavy domain, such as SEQ ID NO: 5 and 6, and any combination of aminoacid distribution or relative codon distribution can be used to replaceX.

In one embodiment, a position denoted with “X” is replaced with an evendistribution of all twenty naturally occurring amino acids except for Cfor CDR1, C and M for CDR2, and C, M, and N for CDR3. For example, “X”in CDR1 is not replaced by C (Cys) residue, “X” in CDR2 is not replacedby C or M residue, “X” in CDR3 is not replaced by C, M, or N residue.Any other combination of amino acids and distribution of each amino acidis deemed equal. Such distributions can be created by DNA degeneratesynthesis methods/technologies such as NNK or TRIM (Ferreira Amaral,Frigotto et al. 2017) (Li A, Sun Z, Reetz M T. 2018) (Suchsland R, AppelB, Müller S. 2018).

Designs of degeneracy within the variable region can be determined asdisclosed herein. Multiple standard methods to design mutations in thevariable regions can also be used to create a more diverse library.Design of the VHO consensus scaffold and/or libraries of degeneracy canalso consider factors such as translation of the VHO scaffold and/orlibraries of degeneracy in bacterial (e.g., Escherichia coli), yeast, ormammalian (e.g., human or Chinese hamster ovary cells). For example,design of the VHO scaffold and/or libraries of degeneracy can considercombinations of codon usage or codon optimization typically designed bythose skilled in the art.

In one embodiment, the polynucleotide sequences in a VHO library asdisclosed herein are placed in a vector such as a bacterial phagemidand/or plasmid. In one embodiment, the vector contains one or moreelements of function for controlled growth via antibiotic resistance, anOri site for DNA replication, an Ori site for phage replication,promoter for driving protein expression, secretion signal (e.g., aleader sequence for driving secretion of translated proteins), and arepressor to control levels of protein expression as typically used bythose skilled in the art. Depending on the host system used, certaincombinations of the codon use and phagemid/plasmid elements can be usedto obtain more efficient expression of the genes into proteins.

In one embodiment, a VHO library disclosed herein is placed into anexpression cassette within a phagemid/plasmid, e.g., pTAVO as shown inFIG. 5(B).

In one embodiment, the present disclosure provides a phage library,wherein the VHO domains as described herein are capable of beingdisplayed or are displayed on a viral particle produced by a prokaryoticor a eukaryotic cell, on a virus that infects a prokaryotic and/oreukaryotic host cell, or on a prokaryotic or a eukaryotic cell.

In one embodiment, the present disclosure provides a VHO library on abacteriophage such as an M13 filamentous bacteriophage. Any coat proteinof a bacteriophage, e.g., M13, can be connected by way of in-framegenetic fusion to either the amino or carboxyl terminal end of a VHOdomain. For example, a VHO can be fused with any coat protein, e.g.,PVII, PIX, PIII, and/or PVI, e.g., pVII and/or pIX, of an M13filamentous bacteriophage. (See Høydahl, Nilssen et al. 2016) The fusioncomprising a VHO and a coat protein can be further fused with other tagor tag combinations understood to those skilled in the art. FIG. 5(A)illustrates a schematic representation showing a VHO fused with abacteriophage coat protein (e.g., VHO fused with coat protein pIX with aHIS tag and HA tag). FIG. 5(C) shows a schematic representation of a VHOfused to an M13 phage (displayed on an M13 phage).

Synthesis and construction of VHO mutational libraries can be conductedby methods understood by one skilled in the art. The VHO mutationallibraries as disclosed herein can be placed in a phagemid/plasmidconstruct by cloning methods understood by one skilled in the art. Forexample, the NcoI and NotI restriction sites depict the area on pTAVO asto where the DNA of the VHO mutational libraries is inserted to obtainligated VHO library constructs such as a VHO phage library. Furthermore,a combination of recombinant and restriction enzyme cloning methods canbe used to construct the VHO mutational libraries within each cassette.

Transformation by VHO Phage Libraries

Ligated VHO phage library constructs can be transformed into cells, suchas MC1061F′ cells or equivalent strains (JM105, JM107, JM110, DH1, GM48,SL10, TD1, MC4100, SK1592, etc.) capable of being infected by M13filamentous bacteriophage, as illustrated by exemplary embodimentsbelow. Transformational efficiencies from 1×10⁶ to 1×10¹¹ (colonies peramount of ligated DNA) can be obtained in a typical laboratoryenvironment. Transformation efficiency is calculated by determining howmany colony forming units (transformants) one can obtain from a reactionof one microgram of DNA.

Bacterial cultures can be transformed with a VHO phage library, e.g., aVHO library with a fixed CDR3 length. If a VHO phage library includesmultiple sub libraries with different CDR3 lengths, multiple bacterialcultures can be transformed respectively with VHO sub-libraries of eachCDR3 length.

Bacterial cultures transformed with a VHO phage library, e.g., a VHOsub-library with a fixed CDR3 length, are grown to log phase. Thecultures are infected with a helper phage, such as a VCSM13 helperphage, and grown overnight under induction conditions. The overnightphage library amplification cultures undergo phage harvest methods.Titers of at least 1×10¹² colony-forming-units or plaque-forming-unitsare obtained. Rolling circle and Sanger sequencing methods are performedon single infected colonies. For example, the sequences obtained fromsequencing of the single infected colonies are processed for translationand then aligned to one another as well as a VHO DNA sequence template(e.g., matching germline Vh sequence obtained from IMGT). This sequenceanalysis looks for in frame translation from the secretion signal to theexpected final stop codon. In one embodiment, any sequence containingCysteine, Methionine, or a stop codon located within one or more of theCDRs is not accepted as an accurate VHO. Typical accuracy of these VHOlibraries range from 30-60%. FIG. 5 (C) portrays an example of how a VHOis displayed on the surface of an M13 phage through a genetic fusionwith the minor coat protein pIX. Fab phage libraries having similardiversities, transformation efficiencies and functional accuracies gaverise to many successful biopanning projects (Shi et al, 2007).

Screening of VHO Phage Libraries

A VHO phage library as disclosed herein can be screened using panning(or biopanning) methods against a target as illustrated by the followingexemplary embodiments. A target can be a protein, cell, tissue, orcombinations thereof. For example, a VHO pIX phage display library isadded to a target for screening. This allows for binding to occurbetween the VHO parts of the phage library and the target. To capturetarget specific VHOs, non-specifically bound VHO pIX phages are washedaway. The VHO-phages still bound to the target after washing arecaptured by either using an acid or just applying bacteria cells fordirect infection. For example, the infected bacteria carrying the VHOcandidates are expanded in culture usually within 4 hours. Once theexpanded culture is at optimal cell density (e.g., OD600 nm=0.6-0.8),helper phage is added to the culture so that the amount of plaqueforming units (pfu) reaches at least 10× greater than the number ofcells. The helper phage infected bacteria cells are induced forexpression of the selected VHO candidates fused to the phage and theculture is incubated overnight. This stage is called phageamplification, which can be performed after each round of biopanningknown by those skilled in the art. For example, the bacterial cellscultures carrying the VHO candidates can be incubated for 8-16 hours.

The steps for biopanning can be repeated for multiple times, such as atleast three times, 4 times, or 5 times. Efficacy as to whetherbiopanning is working is determined by how dense the cultures have grownbetween each round of biopanning since they are under antibioticpressure. The culture from the final round of biopanning is used tocapture the phagemid DNA sequences encoding the selected VHO candidatesfrom the biopanning process, e.g., those VHO candidates that are capableof binding to a target after the biopanning process. The capturedphagemid DNA sequences are used to generate NGS libraries (Suckling,McFarlane et al. 2019) (Head, Komori et al. 2014) so as to sequence eachVHO candidate (Dias-Neto, Nunes et al. 2009). Each panning group, e.g.,a panning group that screens a given target, (for instance a panninggroup focused on human TNF, a panning group focused on cynomolgus TNF,or a panning group focused on mouse TNF), makes up its own NGS ampliconlibrary.

For example, primer-based indices Nextera-XT (www.illumina.com) areapplied in such a way to later distinguish from which panning group eachsequence is selected. The PCR and bead-based purification steps neededto create these indexed amplicon libraries are created based on (16SMetagenomic Sequencing Library Preparation, Illumina part #15044223 RevB.https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf) using 30 ng of panningoutput DNA and 16S primers diluted to 0.1 μM with gene specific sequencepriming sites. Specifically, the forward primer has a TM of 62.6° C. andthe reverse primer has a TM of 61.3° C. Both primers are designed toanneal on flanking areas to capture the CDR diversity of the selectedVHOs from the biopanning process. The final NGS amplicon library productcontains an approximate range of 250-300 bp length region making up ofheavy chain CDR1 through heavy chain CDR3. These amplicon libraries areapplied to a 2×300 paired MiSeq run. Obtained from such a MiSeq run is20-25 million sequences. These sequences are organized by quality scores(Fastq) and separated by biopanning indices utilizing BaseSpace software(Illumina). The data is delivered in unpaired Fastq format separatedaccording to the indices that define each biopanning group. Processingapplications organizes up to twenty-five million sequences down to 1000to 100,000 unique protein sequences per MiSeq run which can further beextrapolated based on indices/biopanning groups as disclosed herein.

Determination of Specificity of Selected VHO Candidates

As an illustrative embodiment, sequence results from NGS are groupedaccording to the targets used in the biopanning or the environment usedin biopanning. For example, the target proteins used can be from threedistinct species: human, cynomolgus monkey (cyno), and mouse. The ECD(extracellular domain) of human TNF shows 97% and 79% identity with theECD of cyno and mouse TNF, respectively. DNA isolated from eachbiopanning group, for example, against human, cyno, and mouse TNF,respectively, is used for creating NGS libraries where each group is“indexed” (Head, Komori et al. 2014) (Li, Zhao et al. 2019) to aid inthe specific recovery of VHO candidate sequences. Initially millions ofsequences are processed into each indexed biopanning group. Within eachbiopanning group there is a sequence distribution of all the selectedVHO candidates. After the DNA sequences of the selected VHO candidatesare processed into peptide sequences, distinct sequences for aparticular panning of the VHO candidates can be determined. For example,specificities such as sequences only found within a biopanning group andsequences found in more than one biopanning group are picked for furtherassessment. A list of candidate sequences from three biopanning groups,i.e., against human, cyno, and mouse TNF, respectively, for furtherassessment is shown in FIG. 6.

The VHO candidates determined from the NGS processing are picked foreither clonal soluble VHO expression or displayed on phage to beassessed for binding specificity to the same respective targets used inthe biopanning process. For example, certain expressed VHO candidates(e.g., with a tag such as HIS or Fc) are applied as analytes in biolayerinterferometry (BLI) kinetics assay. FIG. 7 shows binding activities ofa set of VHO-Fc fusions (VHO3-Fc, VHO4-Fc, and VHO5-Fc, comprising VHOsequences set forth in SEQ ID NOs: 7, 8, and 9, respectively) in BLIexperiments (GatorBio) to two distinct species (human and mouse FCRNproteins) and two different pH specificities (pH 6 and pH 7).

(SEQ ID NO: 7) EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYWMSWVRQAPGKGLEWVSVISGDGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWKGAGRGPGALGGLDVWGQGTLVTVSS. (SEQ ID NO: 8)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVSAIWSDGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRT WGHQFDYWGQGTLVTVSS.(SEQ ID NO: 9) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWVSVINYSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAMGT PGELPLDYWGQGTLVTVSS.

The biopanning, NGS, and binding assay results show whether a VHO, e.g.,a fully human VHO, binds to a target. The VHOs can be displayed on a pIXphage both in a diverse library setting and in the clonal phage stage.The example associated with FIG. 7 demonstrates that VHO phage displaycan be used to generate cross reactive VHO candidates to a target. Thepanning and NGS analysis methods can identify many VHO candidatesequences. More than one VHO candidate shows correlation between thebiopanning and NGS analysis and with the specificity binding results inBLI. The data in FIG. 7 further shows diverse binding activities of theVHOs. For example, the different curves in steps 3 and 4 of the BLI runsare indicative of the diverse binding activities with the use of twoorthologs of the target and two pH values used.

FIG. 8 shows how well behaved (e.g., no severe aggregation) a VHO canbe, compared to the other proteins, such as IgGs. For example, aftercompleting 217 transiently expressed candidates of VHO-Fc fusionproteins, no aggregation was observed with the VHO-Fc fusions. Of the217 candidates, 88% expressed as VHO-Fc dimers had bands thatcorresponded to the MW predicted based on the sequences by non-reducingSDS-PAGE (see examples illustrated in FIG. 8B). FIG. 8A shows examplesof size exclusion chromatography results of two different VHO-Fcmolecules (denoted as “VHO1-Fc” and “VHO2-Fc”) compared to a human IgG1molecule. The size exclusion chromatography chromatograms of FIG. 8A aretypical of VHO-Fc proteins disclosed herein. The single peak profiles onthe chromatogram of FIG. 8A indicate presence of the VHO proteins withthe correct molecular weight with minimal aggregation or clipping.

“VHO1-Fc” and “VHO2-Fc,” characterized in FIG. 8A, comprise thefollowing VHO sequences, respectively.

(SEQ ID NO: 10) EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVSGISYSGSSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARPRWAVPGRGHPRFDYWGQGTLVTVSS. (SEQ ID NO: 11)EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSS FDYWGQGTLVTVSS.

Examples Example 1. Library Construction

An initial cassette containing pIX, HIS, and HA tags without the VHOgene was cloned into pTAVO by using NcoI and Nod restriction enzymesites. FIG. 5(B) illustrates pTAVO with the NcoI and NotI restrictionenzyme sites. CDR1 and CDR2 diversity as DNA fragments synthesized by aCRO were cloned into the pTAVO, which already contains pIX, HIS, and HAtags, by using restriction enzymes such as PstI and NcoI according toappropriate digest parameters. The pTAVO already containing CDR1 andCDR2 diversity, as well as pIX, HIS, and HA tags, was amplified and thenused as a partial library containing vector to insert the more diverseCDR3 region library using restriction enzymes such as PstI and XhoI. T4ligase was used in all cloning steps after gel extraction of vectors andDNA fragments, following the recommended methods from vendors. Theamount of digested CDR1 and CDR2 or CDR3 fragments and digested pTAVO orpartial library pTAVO vectors are placed in an T4 ligase reaction set ata molar ratio of at least 2:1 but not limited to this ratio,respectively. Ligated libraries, e.g., pTAVO containing CDR1, CDR2, andCDR3 diversity, as well as pIX, HIS, and HA tags, were cleaned up in away to avoid DNA loss, for example by glycogen and butanolprecipitation. The ligated libraries at 500-1000 ng of DNA were mixedinto 5-10 aliquots of competent MC1061 F′ E. coli each set at 10⁸ cellsper aliquot. The DNA/cell mixture underwent electroporation followingoptimized parameters for highest transformation efficiencies. Thetransformed cells were allowed to recover from antibiotic resistance,grown to log phase, and then infected with M13 helper phage. Theinfected cells with the ligated libraries were grown under optimal phageand VHO protein expression conditions for 16-18 hours. The VHO libraryphages were precipitated and concentrated 10-fold and stored at −80° C.for use in subsequent biopanning. The culture media used for allbacteria/phage library conditions was 2×YT. Glucose was used to repressVHO expression prior to phage amplification using a Lac repressor. VHOand phage expression were induced by 1 mM IPTG with a Lac promoterwithout glucose.

Example 2. Phage Biopanning

Phage libraries and streptavidin (SA) linked magnetic beads (LifeTechnologies, M280 streptavidin ferrous based beads) were pre-loadedwith biotinylated target antigens, e.g., human, mouse, or cynomolgus TNForthologs. A magnetic separator was used to effectively separate thebeads from solutions during certain steps of the biopanning experiment,e.g., tubes were placed in a magnetic separator during the washing stepsso as to rid non-specific bound phage from the specifically bound phageon the beads. The target bound SA beads were blocked with agents such asmilk or Chemiblocker to reduce non-specific binding by the VHO phagelibraries. Nonspecific (e.g., non-binding to the target-loaded beads)VHO phages were removed by washing with buffer such as PBS. Log phaseMC1061 F′ E. coli cells were used to capture (infect) those phages thatretain the preferred biopanned characteristics, capable of binding tothe target-loaded beads during the last round of biopanning. Afteramplification of the captured VHO phages and subsequent rounds ofbiopanning, the final round of target specific VHO phages captured bylog phase MC1061 F′ by infection was grown overnight under suppressionconditions of expression. DNA preparations on this final culture ofbiopanned output was performed following vendor's protocol.

Example 3. NGS Analysis of Biopanned VHO Candidates

Based on the protocol outlined in the 16S Metagenomic Sequencing LibraryPreparation, Illumina part #15044223 Rev B. documentation(https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf), the following methodswere used to create gene specific NGS amplicon libraries. The finalround of biopanning output DNA was amplified using gene specific forwardand reverse primers, which include the Illumina overhang adaptersequence. PCR was used to perform 25 cycles of amplification using KAPAHiFi Hot Start polymerase, and AMPure XP bead-based PCR cleanup wasperformed on the PCR reaction according to the protocol referencedabove. DNA from the purified PCR reaction was used in a second PCR(index PCR) with 8 cycles of amplification using KAPA HiFi Hot Startpolymerase and AMPure XP bead-based PCR cleanup. The samples were thennormalized, denatured, mixed with denatured PhiX control, and loadedonto a single MiSeq run (Ravi R K, Walton K, Khosroheidari M. 2018) todetermine the sequences of the VHO candidates.

Example 4. VHO Protein Production

VHO sequences established from biopanning and NGS were synthesized andcloned into a mammalian expression vector as Fc fusions (Lo et al,1998). Each VHO-Fc construct was transiently expressed in HEK293 Expicells (Invitrogen). The spent media from a 5-day culture was processedusing a Protein A or Protein G methodology to purify the VHO-Fc protein(Fishman and Berg, 2019).

Example 5. VHO Protein Analysis

VHO-Fc proteins were assessed for yield, purity, and biologic activity.The yield was determined by absorbance at 280 nm using aspectrophotometer. The purity was assessed by SDS-PAGE and themonodispersity was confirmed using size exclusion chromatography. Thebiologic activity was measured by binding to the target protein or tocells with such target on its surface. Binding to a protein target canbe done either by ELISA, biolayer interferometry, or any equivalenttechnique for protein-protein interactions. Binding activity was alsomeasured on cells via flow cytometry.

Example 6. Epitope Screening

A library using a small part of an antibody, the variable heavy region,was designed. This small part allows for more epitopes to be covered ona given target. The VHOs are modularized to capture assorted designs ofCDR diversity to increase the ability to capture more paratopes. Diversemethods of biopanning also improves on generating many epitopes andparatopes. NGS and sequence analysis methods help prioritize whichselected VHOs to express as soluble proteins for downstream activityassessments.

FIG. 9 shows that the percentages of epitopes per candidate screened orcharacterized are higher for the VHOs according to the presentdisclosure, compared to traditional pannings of sdAb, Nb, and VHH.Researchers at Ablynx N.V. conducted a phage display effort against asingle target and were able to get 3-4 epitopes from 96 candidates.(doi:10.3389/fimmu.2017.00420). Thus, the Ablynx study resulted in asuccessful target efficiency of 0.04 or 4% (4 epitope/96 candidates)(doi:10.3389/fimmu.2017.00420). Another group immunized phage displayagainst 5 targets yielding a range of 3-6 epitopes from a range of160-182 candidates. Their successful target efficiency was of 0.03 or 3%(30 epitopes from 867 candidates) (DOI: 10.7554/eLife.48750.009).

A VHO (fully synthetic and fully human) phage display library was usedto pan 5 distinct targets. A total of 31 epitopes to 190 VHO candidateswas obtained, resulting in a successful target efficiency of 0.16 or 16%(31 epitopes from 190 candidates) as shown in FIG. 9. Thus, a VHOlibrary according to the present disclosure had a successful targetefficiency larger than those in the art, which achieved successfultarget efficiencies of less than 5%.

The VHO molecules were expressed in mammalian cells as VHO-aloneproteins and as Fc fusion proteins. The successful targeting VHO hitmolecules in either format also had better expression behavior,monodisperse profiles as shown in FIG. 8, as well as binding activity asshown in FIG. 7.

Further exemplary embodiments are illustrated below.

1. A VHO library of polynucleotides encoding VHO (variable heavy only)domains, wherein the VHO domains have sequence homology and/or canonicalhomology with the Vh domain of a human Vh family.

2. The VHO library of embodiment 1, wherein the human Vh family is ahuman Vh3 family, e.g., IGHV3-23.

3. The VHO library of any of embodiments 1-2, wherein the sequencehomology is at least 75%.

4. The VHO library of any of embodiments 1-3, wherein the VHO domainshave residue diversity throughout the entire regions of the VHO domains.

5. The VHO library of any of embodiments 1-4, wherein the VHO domainshave residue diversity in one or more of the CDR regions.

6. The VHO library of any of embodiments 1-5, wherein the VHO domainscomprise SEQ ID NO: 5:QVQLVESGGGLVKPGGSLRLSCAASGFTFS(X)WVRQAPGKGLEWV(X)DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR(X)WGQGTLVTVSS (SEQ ID NO: 5),wherein position X represents any one of the 20 naturally occurringamino acid residues.

7. The VHO library of any of embodiments 1-5, wherein the VHO domainscomprise SEQ ID NO: 6:

(SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASGFTFS(X)NWVRQAPGKGLEWV(X)DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR(X)WGQGTLVTVS S,wherein position X represents any one of the 20 naturally occurringamino acid residues.

8. The VHO library of any of embodiments 6 and 7, wherein position X isreplaced with an even distribution of all 20 naturally occurring aminoacids except for C for CDR1, C and M for CDR2, and C, M, and N for CDR3.

9. The VHO library of any of embodiments 1-8, wherein the VHO domainshave residue diversity in one or more of the framework regions.

10. The VHO library of any of embodiments 1-9, wherein one or more ofthe framework regions have one or more mutations that provideimprovements in levels of protein expression, protein folding, proteinpurification, binding affinity, downstream target signaling, and/orinhibition of signaling.

11. The VHO library of any of embodiments 1-10, wherein the VHO domainshave residue diversity in one or more of the framework regions and inone or more of the CDR regions.

12. The VHO library of any of embodiments 1-11, wherein the VHO domainshave length diversity in the CDR3 region.

13. The VHO library of any of embodiments 1-11, wherein the lengthdiversity of the CDR3 region ranges from 6 to 30 codons.

14. The VHO library of any of embodiments 1-13, wherein thepolynucleotides encoding VHO domains are inserted into a vector.

15. The VHO library of any of embodiments 14, wherein the vector is aphagemid.

16. The VHO library of any of embodiments 14, wherein the vector is aplasmid.

17. The VHO library of any of embodiments 14-15, wherein the vectorcomprises polynucleotides encoding a coat protein of a bacterial phage.

18. The VHO library of embodiment 17, wherein the bacterial phage is M13bacteriophage.

19. The VHO library of any of embodiments 17-18, wherein the coatprotein is a pVII or pIX coat protein.

20. The VHO library of any of embodiments 14-19, wherein the vectorcomprises one or more tags.

21. The VHO library of embodiment 20, wherein the one or more tags arechosen from polyhistidine (HIS) and hemagglutinin (HA) tags.

22. The VHO library of embodiment 21, wherein the vector comprises apolynucleotide sequence encoding a linker peptide between the VHO andthe one or more tags.

23. The VHO library of embodiment 21, wherein the vector comprises apolynucleotide sequence encoding a linker peptide between VHO and thecoat protein.

24. The VHO library of any of embodiments 22-23, wherein the linkerpeptide is GGGGS (SEQ ID NO: 17).

25. The VHO library of any of embodiments 14-15 and 17-24, wherein thevector comprises elements that are arranged in a manner to allow for theVHO domains to be displayed on a bacteriophage.

26. The VHO library of any of embodiments 14-25, wherein the vectorcomprises elements that are arranged in a manner to allow for the VHOdomains to be expressed.

27. The VHO library of any of embodiments 14, 16, 20-22, 24, and 26,wherein the vector comprises elements that are arranged in a manner toallow for the VHO domains to be expressed without a bacterial phage.

28. The VHO library of any of embodiments 14-27, wherein the vector hasOri site for DNA replication, an Ori site for phage replication, aleader sequence for driving secretion of translated proteins, a promoterfor driving protein expression, and a repressor to control levels ofprotein expression.

29. The vector of any one of embodiments 14-28, wherein the vectorcomprises polynucleotides encoding any one of the VHO domains.

30. A phage library displaying the VHO domains encoded by the VHOlibrary of embodiments 1-28.

31. The phage library of embodiment 30, wherein the VHO domains aredisplayed on a viral particle produced by a prokaryotic or a eukaryoticcell, displayed on a virus that infects a prokaryotic and/or eukaryotichost cell, or displayed on a prokaryotic or a eukaryotic cell.

32. A method of preparing the VHO library of any one of embodiments1-28, comprising: providing polynucleotide sequences encoding the VHOdomains of any one of embodiments 1-13; and inserting the polynucleotidesequences into the vector of any one of 14-29.

33. A method of preparing the phage library of any one of embodiments30-31, comprising: transforming a bacterial cell culture with the VHOlibrary of any one of embodiments 1-28,

allowing the bacterial cell culture to grow to a log phase, infectingthe bacterial cell culture with a helper phage, and

amplifying the bacterial cell culture.

34. A method of identifying a VHO domain of interest that is capable ofbinding a target, comprising: creating a VHO library according toembodiment 32, creating a phage library of according to embodiment 33,and screening the phage library using biopanning against the target toidentify the VHO of interest.

35. The method of embodiment 34, wherein the biopanning step isconducted multiple times.

36. The method of any one of embodiments 34-35, further comprisingsequencing the VHO of interest by NGS.

37. The method of any one of embodiments 34-36, further comprisingevaluating the binding affinity of the VHO of interest to the target.

38. The method of embodiment 37, wherein the binding affinity isevaluated using ELISA.

39. A VHO of interest identified by the method of any one of embodiments34-38.

40. A polypeptide comprising the VHO of embodiment 39, wherein the VHOis fused to a protein chosen from immunoglobulins, receptors, cellsurface proteins, and fragments thereof.

41. A composition comprising the VHO of embodiment 39 or the polypeptideof embodiment 40.

42. A polynucleotide that encodes the VHO of interest of embodiment 39or the polypeptide of embodiment 40.

43. A polypeptide that has greater than 50% identity to the VHO ofinterest of embodiment 39.

44. A polynucleotide that encodes a protein that has greater than 50%identity to the VHO of interest of embodiment 39.

45. A vector comprising the polynucleotides of any one of embodiments 42and 44.

46. A cell comprising the vector of embodiment 29.

47. A cell comprising the vector of embodiment 45.

48. A polypeptide comprising the VHO of embodiment 39, wherein the VHOis genetically fused to any tag, e.g., Fc domain of an immunoglobulinfamily of any species, poly histidine tag, and FLAG tag.

49. A polypeptide comprising the VHO of embodiment 39, wherein thepolypeptide is expressed in mammalian cells.

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We claim:
 1. A VHO library of polynucleotides encoding VHO (variableheavy only) domains, wherein the VHO domains have sequence homologyand/or canonical homology with the Vh domain of a human Vh family. 2.The VHO library of claim 1, wherein the human Vh family is IGHV3-23. 3.The VHO library of claim 2, wherein the VHO domains have residuediversity throughout the entire regions of the VHO domains.
 4. The VHOlibrary of claim 2, wherein the VHO domains have residue diversity inone or more of the CDR regions.
 5. The VHO library of claim 1, whereinthe VHO domains comprise SEQ ID NO: 5: (SEQ ID NO: 5)QVQLVESGGGLVKPGGSLRLSCAASGFTFS(X)WVRQAPGKGLEWV(X)DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR(X)WGQGTLVTVS S,

wherein position X represents any one of the 20 naturally occurringamino acid residues.
 6. The VHO library of claim 1, wherein the VHOdomains comprise SEQ ID NO: 6: (SEQ ID NO: 6)EVQLVESGGGLVQPGGSLRLSCAASGFTFS(X)NWVRQAPGKGLEWV(X)DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR(X)WGQGTLVTVS S,

wherein position X represents any one of the 20 naturally occurringamino acid residues.
 7. The VHO library of claim 5, wherein position Xis replaced with an even distribution of all 20 naturally occurringamino acids except for C for CDR1, C and M for CDR2, and C, M, and N forCDR3.
 8. The VHO library of claim 6, wherein position X is replaced withan even distribution of all 20 naturally occurring amino acids exceptfor C for CDR1, C and M for CDR2, and C, M, and N for CDR3.
 9. The VHOlibrary of claim 2, wherein the VHO domains have residue diversity inone or more of the framework regions.
 10. The VHO library of claim 2,wherein one or more of the framework regions have one or more mutationsthat provide improvements in levels of protein expression, proteinfolding, protein purification, binding affinity, downstream targetsignaling, and/or inhibition of signaling.
 11. The VHO library of claim2, wherein the VHO domains have residue diversity in one or more of theframework regions and in one or more of the CDR regions.
 12. The VHOlibrary of claim 2, wherein the VHO domains have length diversity in theCDR3 region.
 13. The VHO library of claim 2, wherein the lengthdiversity of the CDR3 region ranges from 6 to 30 codons.
 14. The VHOlibrary of claim 2, wherein the polynucleotides encoding VHO domains areinserted into a vector.
 15. The VHO library of claim 14, wherein thevector is a phagemid.
 16. The VHO library of claim 14, wherein thevector is a plasmid.
 17. The VHO library of claim 14, wherein the vectorcomprises polynucleotides encoding a coat protein of a bacterial phage.18. The VHO library of claim 17, wherein the bacterial phage is M13bacteriophage.
 19. The VHO library of claim 18, wherein the coat proteinis a pVII or pIX coat protein.
 20. The VHO library of claim 14, whereinthe vector comprises one or more tags.
 21. The VHO library of claim 20,wherein the one or more tags are chosen from polyhistidine (HIS) andhemagglutinin (HA) tags.
 22. The VHO library of claim 21, wherein thevector comprises a polynucleotide sequence encoding a linker peptidebetween the VHO and the one or more tags.
 23. The VHO library of claim21, wherein the vector comprises a polynucleotide sequence encoding alinker peptide between VHO and the coat protein.
 24. The VHO library ofclaim 22, wherein the linker peptide is GGGGS (SEQ ID NO: 17).
 25. TheVHO library of claim 14, wherein the vector comprises elements that arearranged in a manner to allow for the VHO domains to be displayed on abacteriophage.
 26. The VHO library of claim 14, wherein the vectorcomprises elements that are arranged in a manner to allow for the VHOdomains to be expressed.
 27. The VHO library of claim 14, wherein thevector comprises elements that are arranged in a manner to allow for theVHO domains to be expressed without a bacterial phage.
 28. The VHOlibrary of claim 14, wherein the vector has Ori site for DNAreplication, an Ori site for phage replication, a leader sequence fordriving secretion of translated proteins, a promoter for driving proteinexpression, and a repressor to control levels of protein expression. 29.The vector of claim 14, wherein the vector comprises a polynucleotideencoding any one of the VHO domains.
 30. A phage library displaying theVHO domains encoded by the VHO library of claim
 2. 31. The phage libraryof claim 30, wherein the VHO domains are displayed on a viral particleproduced by a prokaryotic or a eukaryotic cell, displayed on a virusthat infects a prokaryotic and/or eukaryotic host cell, or displayed ona prokaryotic or a eukaryotic cell.
 32. A method of preparing the VHOlibrary of claim 2, comprising: providing polynucleotide sequencesencoding VHO domains; and inserting the polynucleotide sequences avector, wherein the VHO domains have sequence homology and/or canonicalhomology with the Vh domain of human Vh family IGHV3-23.
 33. A method ofpreparing the phage library of claim 30, comprising: transforming abacterial cell culture with a VHO library, allowing the bacterial cellculture to grow to a log phase, infecting the bacterial cell culturewith a helper phage, and amplifying the bacterial cell culture, whereinthe VHO library comprises polynucleotides encoding VHO (variable heavyonly) domains, and wherein the VHO domains have sequence homology and/orcanonical homology with the Vh domain of human Vh family IGHV3-23.
 34. Amethod of identifying a VHO domain of interest that is capable ofbinding a target, comprising: creating a VHO library, creating a phagelibrary of according to claim 33, and screening the phage library usingbiopanning against the target to identify the VHO of interest, whereinthe VHO library comprises polynucleotides encoding VHO (variable heavyonly) domains, and wherein the VHO domains have sequence homology and/orcanonical homology with the Vh domain of human Vh family IGHV3-23. 35.The method of claim 34, wherein the biopanning step is conductedmultiple times.
 36. The method of claim 34, further comprisingsequencing the VHO of interest by NGS.
 37. The method of claim 34,further comprising evaluating the binding affinity of the VHO ofinterest to the target.
 38. The method of claim 37, wherein the bindingaffinity is evaluated using ELISA.
 39. A VHO of interest identified bythe method of claim
 37. 40. A polypeptide comprising the VHO of claim39, wherein the VHO is fused to a protein chosen from immunoglobulins,receptors, cell surface proteins, and fragments thereof.
 41. Acomposition comprising the VHO of claim
 39. 42. A polynucleotide thatencodes the VHO of interest of claim
 39. 43. A cell comprising thevector of claim
 29. 44. A polypeptide comprising the VHO of claim 39,wherein the VHO is genetically fused to a tag selected from Fc domain ofan immunoglobulin family of any species, poly histidine tag, and FLAGtag.
 45. A polypeptide comprising the VHO of claim 39, wherein thepolypeptide is expressed in mammalian cells.